Thakor Devang, Spigelman Igor, Tabata Yasuhiko, Nishimura Ichiro
Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, School of Dentistry, University of California, Los Angeles, California 90095, USA.
Mol Ther. 2007 Dec;15(12):2124-31. doi: 10.1038/sj.mt.6300256. Epub 2007 Jul 10.
Selective modulation of sensory neuron gene expression could have numerous applications for the peripheral nervous system. Here, we report that subcutaneous peripheral injection of plasmid DNA complexed with a non-viral cationized gelatin (CG) vector led to transgene expression in rat lumbar dorsal root ganglia (DRGs). CG/DNA polyplexes appeared to undergo rapid retrograde transport through sciatic and spinal nerves, with reporter gene messenger RNA (mRNA) expression detectable in L4 and L5 DRGs within 60 hours. Maximum transgene expression was observed for polyplexes formed at 7.5:1 CG-to-DNA weight ratio under salt-free conditions, which generated 615 +/- 112 nm nanoparticles with zeta-potential of 9.4 +/- 0.19 mV. Six days after injection of the CG/DNA polypex, reporter gene protein immunofluorescence was observed in 1,164 +/- 176 DRG neurons, representing an estimated transfection rate of 47% of targeted neurons. Reporter gene expression was not detected in heart, lung, or liver tissues, suggesting a lack of systemic uptake. Measurements of tactile sensitivity indicate that CG/DNA injection did not cause behavioral toxicity. The injection platform was further used for plasmid-driven short hairpin RNA-mediated suppression of glyceraldehyde-3-phosphate dehydrogenase. This non-invasive gene delivery system could be used for the mechanistic study and targeted molecular evaluation of peripheral nervous system pathologies such as neuropathic pain.
感觉神经元基因表达的选择性调控在外周神经系统中可能有众多应用。在此，我们报告皮下外周注射与非病毒阳离子化明胶（CG）载体复合的质粒DNA可导致大鼠腰段背根神经节（DRG）中的转基因表达。CG/DNA多聚体似乎通过坐骨神经和脊髓神经进行快速逆行运输，在60小时内可在L4和L5背根神经节中检测到报告基因信使核糖核酸（mRNA）表达。在无盐条件下，以7.5:1的CG与DNA重量比形成的多聚体观察到最大转基因表达，其产生了615±112纳米的纳米颗粒，ζ电位为9.4±0.19毫伏。注射CG/DNA多聚体六天后，在1164±176个背根神经节神经元中观察到报告基因蛋白免疫荧光，估计靶向神经元的转染率为47%。在心脏、肺或肝脏组织中未检测到报告基因表达，表明没有全身摄取。触觉敏感性测量表明，CG/DNA注射未引起行为毒性。该注射平台还被用于质粒驱动的短发夹RNA介导的甘油醛-3-磷酸脱氢酶抑制。这种非侵入性基因递送系统可用于周围神经系统疾病如神经性疼痛的机制研究和靶向分子评估。
